Several publications and patent documents are cited throughout this application in order to better describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein.
Engineering the plastid genome (ptDNA) rather than the nuclear genome gives rise to high protein expression levels and facilitates transgene containment. Plastid transformation involves targeted insertion of the transforming DNA into the plastid genome (ptDNA) by homologous recombination and amplification of the rare, transformed copies by selection for antibiotic resistance encoded in the vector (Bock, 2001; Staub, 2002; Maliga, 2004). Selection for vector-encoded antibiotic resistance genes is essential to obtain uniform transformation of the 1000 to 10000 ptDNA copies present in a higher plant. Following selection, continued expression of the selectable marker gene places an unnecessary metabolic burden on the plant, interferes with the need to use the same marker gene for multistep engineering and reduces consumer acceptance of the transgeneic plant. Thus, once transformation of ptDNA is accomplished, it is often desirable to eliminate the marker gene (Maliga, 2004).
Accordingly, it is an object of the invention to provide nucleic acid constructs and methods of use thereof for selective removal of predetermined sequences from the plastid genome.